Human parainfluenza virus type 3 (HPIV3) is an important respiratory pathogen of infants, young children, elderly and immunocomprimised individuals for which an effective vaccine or treatment is not currently available. The goals of this proposal are to gain a thorough understanding of the viral mechanisms of RNA transcription and replication, and to develop a system to analyze the assembly/release and attachment/entry steps of the HPIV3 replicative cycle. A minigenome reverse genetics system, recently developed for HPIV3, will be used to delineate the sequences involved in promoting HPIV3 RNA replication and transcription (specific aims 1 and 2). The first aim involves analysis of a large RNA region of questionable involvement in replication, with the goal of unequivocally defining the significance of this region in replication. The second aim seeks to determine whether a particular sequence serves as part of the promoter for transcription, thereby distinguishing a promoter for transcription from a promoter for replication. Specific aim 3 seeks to develop the established minigenome system for HPIV3, which allows analysis RNA replication and transcription, into a system in which virus-like particles can be produced. This will allow for functional analysis of the assembly/release and attachment/entry steps of the viral life cycle in a convenient, efficient manner. Establishing such a system will allow for future study of the protein-protein interactions involved in these processes, and could thereby provide target interactions for drug development.